Chlamydia trachomatis suppresses host cell store-operated Ca2+ entry and inhibits NFAT/calcineurin signaling

The obligate intracellular bacterium, Chlamydia trachomatis, replicates within a parasitophorous vacuole termed an inclusion. During development, host proteins critical for regulating intracellular calcium (Ca2+) homeostasis interact with the inclusion membrane. The inclusion membrane protein, MrcA, interacts with the inositol-trisphosphate receptor (IP3R), an ER cationic channel that conducts Ca2+. Stromal interaction molecule 1 (STIM1), an ER transmembrane protein important for regulating store-operated Ca2+ entry (SOCE), localizes to the inclusion membrane via an uncharacterized interaction. We therefore examined Ca2+ mobilization in C. trachomatis infected cells. Utilizing a variety of Ca2+ indicators to assess changes in cytosolic Ca2+ concentration, we demonstrate that C. trachomatis impairs host cell SOCE. Ca2+ regulates many cellular signaling pathways. We find that the SOCE-dependent NFAT/calcineurin signaling pathway is impaired in C. trachomatis infected HeLa cells and likely has major implications on host cell physiology as it relates to C. trachomatis pathogenesis.


Results
Store-operated Ca 2+ entry is impaired by mid-cycle of the C. trachomatis developmental cycle. To investigate the impact of chlamydial infections upon intracellular Ca 2+ mobilization, the ratiometric Ca 2+ indicator, Fura-2, AM was used to assess changes in host cell intracellular Ca 2+ concentration ([Ca 2+ ] i ). HeLa cells either uninfected or infected with C. trachomatis L2 were loaded with Fura-2, AM at the desired time post infection. The binding of Ca 2+ to Fura-2, AM induces a shift in its fluorescence excitation from 380 to 340 nm. Therefore, a 340 nm/380 nm fluorescence ratio of Fura-2, AM was used to determine relative changes in Ca 2+ concentration. A Ca 2+ re-addition assay 27 was used to obtain ratiometric measurements in a resting state, during induced ER Ca 2+ leakage, and throughout SOCE. Following a resting state baseline reading in a Ca 2+ -free Ringer's solution, cells were incubated with Ca 2+ -free Ringer's solution containing either thapsigargin (TG) or the vehicle control, DMSO. TG is an inhibitor of the SERCA Ca 2+ pump responsible for mobilizing Ca 2+ from the cytosol into the lumen of the ER or sarcoplasmic reticulum. TG thus causes an increase in cytosolic Ca 2+ by impairing ER Ca 2+ uptake while ER Ca 2+ depletion occurs via passive ER Ca 2+ leakage through ER translocon complexes 28,29 . When TG treated and DMSO control HeLa cells were moved to a Ca 2+ -containing Ringer's solution, a distinctive increase in [Ca 2+ ] i was detected in TG-treated, but not DMSO treated, cells indicating that Ca 2+ depletion of the ER resulted in the activation of SOCE (Fig. 1a). A STIM1 siRNA knockdown was performed to verify this methodology (Extended Data Fig. 1).
To interrogate changes in Ca 2+ mobilization during C. trachomatis development, the Fura-2, AM Ca 2+ readdition assay was performed with HeLa cells infected with C. trachomatis L2 at early-(1.5 hpi), early-to-mid-(8 hpi), mid-(24 hpi), and late-(46 hpi) cycle developmental time points (Fig. 1a). TG induced a significant increase in cytosolic Ca 2+ at all time points for uninfected and infected cells, except for 46 hpi, indicating TG triggered ER Ca 2+ egress in infected and uninfected cells (Fig. 1b). While the SOCE peak was not significantly different between infected and uninfected cells at 1.5 hpi or 8 hpi, it was significantly depressed in infected cells at the mid-cycle time point (24 hpi) and the 46 hpi time point, indicating C. trachomatis impairs SOCE of the host cell by a mid-cycle developmental timepoint (Fig. 1c). At 46 hpi, the baseline level of F340/F380 was elevated in the infected vs the uninfected. The likely interpretation of this would be that membranes were compromised due to lysis resulting in elevated [Ca 2+ ] i at this timepoint which is near the end of the C. trachomatis developmental cycle, and may explain why TG-induced ER Ca 2+ egress and SOCE at this time point were impaired. Intracellular pathogens depend upon viability of the host cell to complete their intracellular development 2 . We reconfirmed the viability of C. trachomatis infected cells at each of the time points analyzed. There was no significant difference in viability of uninfected versus infected cells at 8 h or 24 h post-infection. Viability was 89.3% ± 1.6% in uninfected versus 91.5% ± 2.4% (Mean ± SD; n = 6) in infected cells at 8 h post-infection and 90.8% ± 2.7% in uninfected versus 93.7% ± 2.0% in infected cells (Mean ± SD; n = 6) at 24 h post-infection. Viability dropped slightly in infected cells at 46 h post-infection from 88.0% ± 3.3% (Mean ± SD; n = 6) in uninfected to 80.7% ± 1.9% (Mean ± SD; n = 6) in infected cells (p < 0.013).
Verification of C. trachomatis-suppressed SOCE using single-cell analysis. The Fura-2, AMbased method measured changes in cytoplasmic Ca 2+ at the cell population level. To assess the influence of C. trachomatis serovar L2 on host cell Ca 2+ mobilization at a single cell level, we used the Ca 2+ indicator Fluo-4, AM with live-cell microscopy to determine changes in [Ca 2+ ] i in infected and uninfected cells. To quantify the normalized relative change in Fluo-4, AM fluorescence intensity (F) for each cell, ΔF/F 0 was calculated. The baseline resting state fluorescence (F 0 ) was the average of the first four mean intensity measurements in Ca 2+ -free Ringer's solution, and ΔF = F − F 0 was used to calculate the change in fluorescence.
Analysis of [Ca 2+ ] i mobilization in uninfected and C. trachomatis-infected cells was performed at a mid-cycle (24 h) developmental timepoint. In uninfected and infected HeLa cells treated with the DMSO carrier, stochastic Ca 2+ elevations were observed in a small subset of cells, and when DMSO-treated control and infected cells were placed in Ca 2+ -Ringer's solution, there was a modest increase in [Ca 2+ ] i (Fig. 2a). When cells were treated with TG, a dramatic increase in ΔF/F 0 was observed for both uninfected and infected cells, indicating the TG Ringer's solution resulted in a striking increase in ΔF/F 0 associated with SOCE in uninfected but not infected cells (Fig. 2a). The mean of the single-cell measurements provides a visualization of the single cell Ca 2+ flux trends for each condition (Fig. 2b). TG-treated, C. trachomatis-infected cells demonstrated a significant increase in relative fluorescence of the Fluo-4, AM indicator compared to uninfected TG-treated cells (Fig. 2c). At 44 hpi, there was no significant difference in the TG peak between TG-treated uninfected and infected cells. (Extended Data Fig. 2a-c). These results indicate that TG induces Ca 2+ egress from the ER of uninfected and infected cells. The SOCE of uninfected and infected cells was also assessed using the Fluo-4, AM indicator. The mean ΔF/ F 0 at the SOCE peak for HeLa cells infected with C. trachomatis serovar L2 was severely reduced compared to uninfected cells. Furthermore, 53% of the uninfected, TG-treated cells had a ΔF/F 0 greater than 0.5 compared with only 3% infected cells (Fig. 2d). Additionally, uninfected and infected DMSO treated cells had 5% and 2% of cells, respectively, with a ΔF/F 0 of greater than 0.5 (Fig. 2d). At 44 hpi, infected cells had a significantly reduced ΔF/F 0 mean at the SOCE peak compared to uninfected, and only 3% of the cells had a SOCE peak greater than 0.5 in the infected and TG-induced condition compared to 37% in the uninfected and TG-induced condition (Extended Data Fig. 2d). Collectively, the Fluo-4, AM single-cell analysis demonstrated that SOCE of the host cell is impaired by mid-cycle and remains suppressed at later developmental time points.  The peak Ca 2+ efflux from the ER induced by TG for each time point was measured. The TG treatment was compared to DMSO for either uninfected or infected cells. Student's T-test was used to compare the DMSO to the TG treatment, n = 3. (c) Peak SOCE for each time point was calculated. Student's T-test was used to compare the uninfected TG treated condition to the infected TG treated conditions, n = 3. The SOCE peak for TG-treated samples was not significantly different between infected and uninfected cells at 1.5 hpi (p = 0.0929) or 8 hpi (p = 0.3491), however, it was significantly reduced in infected cells at the mid-cycle time point (24 hpi) (p = 0.002) and the 46 hpi time point (p = 0.0012). Data are presented as mean ± SEM.  30 . Utilizing GCaMP6m, a relative change in GFP fluorescence can be used to determine changes in [Ca 2+ ] i . C. trachomatis L2 expressing mScarlet permitted visualization of chlamydial inclusions during imaging. HeLa cells infected with mScarlet C. trachomatis L2 and transfected with pN1-GCaMP6m-XC were tested to assess Ca 2+ mobilization. The single-cell and mean analysis of the relative fluorescence change of GCaMP6m demonstrated similar trends as the Fluo-4 analysis with SOCE of infected cells impaired relative to uninfected cells ( Fig. 3a, b). The single-cell analysis at the TG peak indicated that both uninfected and infected cells had elevated fluorescence upon TG addition compared to the DMSO vehicle control (Fig. 3c). The single-cell analysis of the SOCE peak indicated that uninfected cells treated with TG had an increased relative change in GCaMP6m fluorescence compared to the DMSO control, however, there was no significant difference in the SOCE peak between the 24 hpi cells treated with DMSO and TG (Fig. 3d). The GCaMP6m analysis of HeLa cells at a mid-to-late developmental cycle timepoint, 36 hpi, with C. trachomatis serovar L2 demonstrated that SOCE was suppressed in infected cells (Extended Data Fig. 3). The Fura-2, Fluo-4, and GCaMP6m Ca 2+ indicators collectively demonstrated that SOCE is impaired in HeLa cells infected with C. trachomatis L2 by mid-cycle developmental time point.

Genetically encoded Ca 2+ indicator confirms SOCE inhibition in
To determine if a urogenital C. trachomatis serovar also impairs SOCE of the host cell, the Ca 2+ re-addition assay was performed using GCaMP6m in HeLa cells infected with C. trachomatis serovar D at a chlamydia developmental midpoint (24 h). Similar to the C. trachomatis L2 results, C. trachomatis D impaired SOCE of the host cell by the mid-cycle developmental timepoint (Extended Data Fig. 4). 2+ ] i caused by SOCE result in the activation of various Ca 2+ -dependent pathways. To gain insights into physiological consequences of impaired SOCE in C. trachomatis infected cells, a specific SOCE-dependent pathway was investigated. Sustained elevated [Ca 2+ ] i activates the calcineurin-NFAT signaling pathway via the Ca 2+ -mediated binding of calmodulin to calcineurin, the calcineurin-dependent dephosphorylation of the NFAT transcription factor to expose its nuclear localization signal, and the subsequent cytosol-to-nucleus translocation of NFAT. We investigated the SOCE-induced nuclear localization of NFAT1 in C. trachomatis-infected cells at the mid-cycle time point to determine if a SOCE-dependent pathway is abrogated during C. trachomatis infection. HeLa cells infected with mScarlet-expressing C. trachomatis L2 were transfected with the HA-NFAT1(4-460)-GFP plasmid. A pilot study indicated that the optimal time following TG treatment and Ca 2+ incubation for NFAT-GFP . Therefore, imaging for NFAT-GFP nuclear translocation was performed immediately following TG or DMSO treatment, and again at 18 min post-Ca 2+ incubation. Imaging of NFAT-GFP expressing HeLa cells treated with DMSO demonstrated no noticeable change in nuclear NFAT-GFP fluorescence following the 18 min Ca 2+ incubation, while the TG treatment caused a dramatic increase in nuclear NFAT-GFP following the Ca 2+ incubation (Fig. 4a). However, neither the DMSO nor TG treatment caused a noticeable increase of nuclear NFAT-GFP in C. trachomatisinfected cells at 24 hpi (Fig. 4a). A representative time course video of NFAT-GFP-expressing HeLa cells infected with mScarlet C. trachomatis L2 did not demonstrate NFAT-GFP nuclear localization in the presence of TG, however, NFAT-GFP-expressing cells in the same field of view without a chlamydial inclusion showed nuclear translocation of NFAT-GFP (Supplementary Video 2). Image analysis was performed to calculate the NFAT-GFP nuclear-to-cytoplasmic fluorescence ratio (N/C) for each condition. The NFAT-GFP N/C was measured as the mean fluorescence intensity of nuclear NFAT-GFP / the mean fluorescence intensity of cytoplasmic NFAT-GFP for individual cells. Cells with an observable inclusion containing mScarlet C. trachomatis were calculated for infected cells. The only significant difference identified between the pre-and post-incubation with Ca 2+ -containing Ringer's solution was for the uninfected + TG condition, which demonstrated a substantial increase in the NFAT-GFP N/C ratio following the 18 min Ca 2+ incubation (p value < 0.0001). No significant difference in NFAT-GFP N/C was observed for C. trachomatis-infected cells treated with TG post-Ca 2+ incubation. (Fig. 4b). To visualize how the NFAT-GFP N/C ratio changed from post-TG or -DMSO treatment to post-Ca 2+ incubation for each cell, a percent change in NFAT-GFP N/C ratio was calculated per cell (Fig. 4c). Collectively, the NFAT-GFP N/C ratio assessment demonstrated a severe reduction in NFAT-GFP nuclear localization in C. trachomatis L2 infected HeLa cells when induced to undergo SOCE.

Discussion
We examined Ca 2+ dynamics in C. trachomatis-infected cells and demonstrate that C. trachomatis inhibits SOCE of the host cell by a mid-cycle (24 h) developmental time point. This inhibition of SOCE was confirmed using three independent methods of intracellular Ca 2+ quantitation. High concentrations of intracellular Ca 2+ resulting from SOCE induction can act as a second messenger to activate numerous signaling pathways 18,31 . Among the pathways activated is the calcineurin/NFAT pathway. Sustained high concentrations of intracellular Ca 2+ www.nature.com/scientificreports/ activates the phosphatase calcineurin which, in turn, dephosphorylates the cytoplasmic components of the NFAT transcription complex to trigger NFAT translocation into the nucleus 32 . NFAT transcription complexes regulate genes encoding immunomodulatory proteins or involved in developmental cellular differentiation 19,33,34 . C. trachomatis inhibition of SOCE had the downstream effect of inhibiting the calcineurin/NFAT pathway, thus providing a biological confirmation of the intracellular Ca 2+ quantitation. The impairment of NFAT1 nuclear translocation demonstrate a specific signaling pathway that is affected by suppressing SOCE in C. trachomatis infected cells and likely has a multifaceted impact on host cell physiology and chlamydial pathogenesis. At the end of their developmental cycle, chlamydiae are released by one of two distinct mechanisms for infection of adjacent cells and subsequent cycles of infection. Infectious EBs are released either by lysis of the infected cell, or intact or partially intact membrane bound inclusions are released by a process known as extrusion 35 . The cellular requirements for the two different release mechanisms are unique. Myosin II and Ca 2+ are essential for chlamydial extrusion-based dissemination 14,17,35 . Ca 2+ appears to be an important determinant for chlamydial release mechanism from the host cell, however, the suppression of SOCE indicates that other sources of Ca 2+ must be utilized. The lack of synchrony and the fact that all infected cells do not undergo extrusion may present challenges in characterizing the formation of Ca 2+ microenvironments near the inclusion microdomain.
Although the detailed mechanisms of chlamydial inhibition of SOCE remain unknown, at least two host components involved in calcium homeostasis are inappropriately localized in C. trachomatis infected cells. STIM1 and IP 3 R are recruited to and enriched in microdomains on the inclusion membrane 17,26 . IP 3 R is recruited to the inclusion membrane through interactions with the chlamydial protein, MrcA 17 . ATP-induced IP 3 R activation in C. trachomatis infected cells 36,37 suggests MrcA does not have an inhibitory effect on IP 3 R. The mechanism of STIM1 recruitment is unknown though STIM1 is known to form complexes with IP 3 R 38 . Although STIM1 is recruited to the chlamydial inclusion, Orai1 was not and remained localized to the host plasma membrane 26 . A model for possible interactions and events associated with inhibition of SOCE and NFAT translocation is depicted in Fig. 5.
TG treatment induces puncta formation and co-localization of Orai1 and STIM1 in uninfected cells 39 . Puncta formation was previously observed in cells overexpressing fluorescent protein tagged STIM1 and Orai1 26 . While www.nature.com/scientificreports/ different pools of mCherry-STIM1 localized to the inclusion or the ER, Orai1-GFP did not associate with the inclusion membrane. When these cells were induced with TG to undergo SOCE, the inclusion membrane-associated mCherry-STIM1 remained at the inclusion, but the pool of ER-distributed mCherry-STIM1 demonstrated punctate staining indicative of ER-PM junctions necessary for the formation of CRAC channels. This suggested that an unknown chlamydial factor retains STIM1 at the inclusion membrane when CRAC channel formation is induced and likely limits the amount of available STIM1 for formation of these channels. Some caution must be exercised in interpretation of these results since overexpression of STIM1 or Orai1 can dramatically increase the number of CRAC channels 40 . The stoichiometry of STIM1 to the C. trachomatis sequestering agent will thus be important when assessing the influence of C. trachomatis on SOCE since STIM1 binding to the inclusion membrane presumably lowers the available STIM1 for binding Orai1 and could lower the endogenous STIM1:Orai1 molecular ratio. Re-analysis of the data from Dzakah 41 revealed that none of the isoforms of STIM1 or Orai1 were significantly up-or down-regulated at 20 h post chlamydia infection. As to whether or not STIM1 expressed at endogenous levels are able to form puncta in infected cells remains unclear. The primary sites of C. trachomatis infection for genital infections in women and ocular infections are the endocervical epithelium and conjunctival epithelium, respectively 4 . As the first targets of infection, mucosal epithelial cells act as first responders to pathogen challenge by the secretion of cytokines and chemokines, and thus play a key role in the innate immune response 42 . In an immortalized primary human endocervix-derived epithelial cell line, a productive C. trachomatis infection mitigated a pro-inflammatory cytokine and chemokine response. Although the mechanism was not defined, it was proposed that the circumvention of a robust cytokine and chemokine response represented a potential evasion strategy promoting the establishment of a favorable intracellular niche within the endocervix epithelium 43 . SOCE impacts cytokine and chemokine signaling events 31 . The influence of C. trachomatis-mediated suppression of SOCE warrants further investigation.
The impairment to calcineurin activation and NFAT1 nuclear translocation demonstrates a specific signaling pathway that is affected by chlamydial suppression of SOCE. Although the major functions of NFAT are often considered in innate immune cells such as lymphocytes, macrophages, dendritic cells, or neutrophils, NFAT plays multiple additional roles in development and cellular differentiation 19 . NFAT is also expressed in multiple cell types 19 , including epithelial cells [44][45][46] and endothelial cells 47 , however, direct information on how disruption of NFAT signaling might impact pathogen interaction with epithelial cells is limited. African swine fever virus inhibits NFAT-regulated transcription of immunomodulatory proteins by synthesis of a protein, A238L, that directly binds to and inhibits the calcineurin phosphatase activity required for NFAT activation 48 . Reactivation of latent Epstein-Barr virus to a lytic state via a Ca 2+ /calcineurin dependent activation of NFAT by a complex mechanism has been proposed to represent a negative feedback loop involving a viral protein, Zta, that directly binds to and www.nature.com/scientificreports/ attenuates NFAT 49 . H. pylori has the capacity to influence NFAT activity either positively or negatively by the bacterial proteins CagA or VacA, respectively, in gastric epithelial cells 46 . VacA forms an anion selective channel in the plasma membrane 50 , which is believed to deregulate membrane depolarization and inhibit SOCE. Although the mechanism of SOCE inhibition differs, C. trachomatis also inhibits SOCE and subsequent NFAT nuclear translocation. NFAT is a transcriptional regulator that, upon translocation to the nucleus, induces expression of several genes important in innate immune responses 51 . Presumably, inhibition of SOCE by chlamydiae and the resultant inhibition of NFAT translocation blocks transcription of NFAT regulated genes. We hypothesize that this inhibition of SOCE and NFAT signaling promotes chlamydial survival, possibly by downregulation of chemokine or cytokine production. Further studies in human primary cervical epithelial cells or animal model systems will undoubtedly be necessary to fully elucidate the benefits to chlamydial and host survival.

Methods
Bacterial and mammalian cell culture. HeLa 229 human cervical epithelial-like cells (American Type Culture Collection) were cultivated in RPMI-1640 (Gibco) supplemented with 5% fetal bovine serum (HyClone) at 37 °C and 5% CO 2 in a humidified incubator. The C. trachomatis D (UW-3-Cx) and L2 (LGV 434) serovars were cultured in HeLa cells and EBs purified by density gradient centrifugation as previously described 52  www.nature.com/scientificreports/ cence measurements were performed as described in the Fluo-4, AM Ca 2+ measurements section. Imaging was performed at 37 °C, 5% CO 2 , 92% relative humidity using a stage-top incubation chamber (Okolab).

NFAT1-GFP nuclear translocation assay. Infections and transfections were performed as stated in the
GCaMP6m Ca 2+ measurement section, except that cells that were transfected with the HA-NFAT1(4-460)-GFP plasmid. The HA-NFAT1(4-460)-GFP plasmid was a gift from Anjana Rao (Addgene plasmid # 11,107 ;; RRID:Addgene_11107) 55 . At the desired time post infection, cells were washed twice with PBS, and then incubated in Ca 2+ -free Ringer's solution containing 1 ug/mL Hoechst stain for 30 min. Buffer was then exchanged with either DMSO or 2 µM TG Ca 2+ -free Ringer's solution. Immediately following treatment, an image was acquired using the Nikon Ti2e with a CFI60 Super Plan Fluor Phase Contrast ADM ELWD 40 × Objective Lens. ND acquisition was programmed to acquire images from 4 locations per well for each condition. After incubating cells with either DMSO or TG for 5 min, the solution was exchanged with the Ca 2+ -containing Ringer's solution, and then imaged at 18 min post Ca 2+ addition. Imaging was performed at 37 °C, 5% CO 2 , 92% relative humidity using a stage-top incubation chamber (Okolab). Following imaging, Huygens Essential software version 20.04 (Scientific Volume Imaging) was used to stabilize frames between time points and Imaris × 64 software version 9.6.0 (Oxford Instruments) was used to measure the mean fluorescence intensity of NFAT-GFP in the nucleus and the cytoplasm of individual cells before and after SOCE induction. The NFAT-GFP N/C ratio was calculated as the mean nuclear NFAT-GFP fluorescence intensity ∕ the mean cytoplasmic NFAT-GFP fluorescence intensity. . Expression levels were calculated for each gene simultaneously with ambiguously mapped reads counting as partial matches and transcripts were compared across all samples normalizing to the median gene expression ratios using the DESeq2 method. Significant differences in expression were determined as greater than 1 or less than − 1 log2 fold change with a p-value of < 0.05.

Statistics
Statistical analysis was conducted using Prism version 9.1.1 software for Windows (GraphPad). An unpaired Student's T test was performed for Fura-2, AM analysis, and Kruskal-Wallis test with Dunn's posttest was performed for Fluo-4, AM relative change, GCaMP6m relative change, and NFAT-GFP N/C ratios. The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.